Ionic reservoir through application of an electrical potential

ABSTRACT

An improved membrane based biosensor incorporates sensing and reference electrodes and a dc electrical potential produced by a counter electrode. The biosensor incorporates ionophores. The conductivity of the membrane is dependent on the presence or absence of an analyte. A functional reservoir exists between the sensing electrode and a lipid membrane deposited on the sensing electrode. The invention also includes the method of detecting the presence or absence of the analyte by use of the biosensor.

The present invention relates to an improved membrane based biosensorand to a method of improving the performance of membrane basedbiosensors.

Biosensors based on ion channels or ionophores contained within lipidmembranes that are deposited onto metal electrodes, where the ionchannels are switched in the presence of analyte molecules have beendescribed in International Patent Application Nos. WO 92/17788, WO93/215289, WO 94/07593 and U.S. Pat. No. 5,204,239 (the disclosures ofwhich are incorporated herein by reference). As these biosensors rely onchanges in ion conduction through the membrane, usually mediated by anionophore, it is important that there exists an ionic reservoir betweenthe electrode and the lipid membrane. Ideally this ionic reservoirbetween the electrode and the lipid reservoir is not totally depleted orfilled, by conduction through the ionophore, during the course of themeasurement cycle. The usual method of measuring the conductance changesis the use of alternating current (AC) impedance spectroscopy. Theabovementioned disclosures have shown that good reservoirs can beproduced using special linker lipid compounds.

The present inventors have now found that the application of a directcurrent (dc) potential offset superimposed onto the AC impedance signalcan influence the apparent conduction of ions by the ionophore throughthe membrane. Without wishing to be bound by scientific theory it isbelieved that this modification of the ionophore conduction occursthrough the modulation of the reservoir capacity and improvement in thereservoir homogeneity. This improvement in conduction of ions by theionophore therefore allows the use of less ionophore which may be usefulin producing more sensitive sensor membranes as less analyte is requiredto switch the ionophore on/off. A negative dc potential applied to themetal electrode has been shown to improve the ion conduction byionophores, whereas a positive dc potential applied to the metalelectrode has been shown to decrease and even negate the apparentconduction of the ionophores through the membrane. This effect isespecially noticeable when membranes are formed containing phosphatidylcholine based lipids. The inventor has found that by controlling the dcoffset, the reproducibility of the ionophore conduction is greatlyimproved.

Accordingly, in a first aspect the present invention consists in animproved membrane based biosensor comprising a lipid membraneincorporating ionophores the conductivity of the membrane beingdependent on the presence or absence of an analyte, a referenceelectrode, a sensing electrode onto which is deposited the lipidmembrane such that a functional reservoir exists between the lipidmembrane and the sensing electrode, the improvement comprising includingin the biosensor means to apply a dc electrical potential offset to thesensing electrode relative to the reference electrode.

In a second aspect the present invention consists in an improved methodof detecting the presence or absence of an analyte in a sample using amembrane based biosensor comprising a lipid membrane incorporatingionophores the conductivity of the membrane being dependent on thepresence or absence of the analyte, a reference electrode, a sensingelectrode on to which is deposited the lipid membrane such that afunctional reservoir exists between We lipid membrane and the sensingelectrode, the improvement comprising applying a dc electrical potentialoffset to the sensing electrode relative to the reference electrode.

In a third aspect by incorporating ionisable, polarisable, dipolar orotherwise electroactive species within the membrane based biosensorcomprising a lipid membrane incorporating ionophores the conductivity ofthe membrane being dependent on the presence or absence of an analyte. areference electrode, a sensing electrode onto which is deposited thelipid membrane such that a functional reservoir exists between the lipidmembrane and the sensing electrode, the appropriate dc potential can beinduced between the sensor electrode and the analyte solution

Although it is envisaged that generally it is preferred to apply anegative potential onto the metal sensor electrode in order to improvethe ionophore conduction, it may be useful in some circumstances toapply a positive potential onto the metal sensor electrode thus reducingor negating the apparent ionophore conduction through the membrane.

In a preferred embodiment of the present invention a dc potential ofbetween +500 mV to −500 mV is applied to the sensing electrode.

In a further preferred embodiment the dc offset is produced through theuse of a counter electrode where the electrochemical potential betweenthe counter electrode and the sensing electrode produces an electricalpotential of between 0 to −500 mV, with the sensing electrode being atthe negative potential.

In a preferred embodiment the counter electrode is made from stainlesssteel.

In a further preferred embodiment the counter electrode is made fromtitanium.

In a further preferred embodiment the counter electrode is made fromsilver, gold, platinum, palladium, copper, chromium or molybdenum.

In another preferred embodiment the counter electrode is made frommetals that are capable of being deposited in a thin film onto aplastic, glass or silicon substrate, said metals being stable for atleast 30 minutes in aqueous solution and sets up the appropriateelectrochemical potential relative to the sensing electrode on additionof an aqueous solution.

In a further preferred embodiment of the present invention the counterelectrode is an electrochemically neutral metal relative to the sensingelectrode and the dc electrical potential of between +500 to −500 mV iscreated by electronic means.

In a further preferred embodiment of the present invention the counterelectrode produces an electrochemical potential relative to the sensingelectrode which is enhanced or negated or reversed using a dc electricalpotential created by electronic means to give a potential of between+500 to−500 mV.

In yet another preferred embodiment of the present invention, the dcoffset potential of the sensing electrode, onto which is deposited thelipid membrane, is controlled using a three terminal measurement, wherethe impedance measurement is made between the counter electrode and theworking electrode which is the sensing electrode and where the dc offsetpotential is controlled by a reference electrode to be between +500 to−500 mV as required.

The metals used for the counter electrode and the reference electrode inthe three terminal measurement may be any of the commonly used metalsand electrode combinations commonly used in these measurements as knownto those skilled in the art.

In a further preferred embodiment of the present invention the metalused for the sensing electrode is a layer of freshly evaporated orsputtered gold. Alternatively, a freshly cleaned gold surface, which canbe produced using plasma etching or ion-beam milling, can be used.

It is further preferred that the first layer of the lipid membrane isproduced using the linker lipid shown in FIG. 1, the disulfide ofmercaptoacetc acid, linker gramicidin shown in FIG. 2, the membranespanning lipid (C) and the membrane spanning lipid (D) both shown inFIG. 3.

It is further preferred that the second layer of the lipid membrane isproduced from diphytanyl phosphatidyl choline, glycerol diphytanylether, shown in FIG. 7. and biotinylated gramicidin shown in FIG. 4.

In a further preferred embodiment the second layer lipid contains atleast a proportion of a phosphatidyl choline, or phophatidylethanolamine or phosphatidic acid lipid.

In a further preferred embodiment the second layer lipid contains atleast a proportion of a charged lipid.

In a further preferred embodiment the lipid membrane is a monolayer.

As will be appreciated by those skilled in the art, if the sensing of ananalyte occurs through the swing off or on of an ionophore containedwithin the lipid sensing membrane on addition of nalyte, then it ispossible to monitor this change in conduction by measuring the amount ofelectrical potential required in order to maintain the membraneconduction value at the initial ungated membrane conduction value. Themagnitude and sign of the electrical potential is then related to theamount of analyte present in the sample.

By increasing the signal spectral inhomogeneity the information contentin the sign can be increased with the consequent possibility of improvedsignal to noise. One mechanism for achieving this is to take advantageof the system voltage dependence by applying a non sinusoidal excitationand then analyzing the results by fourier transform in which case thesignal information content will be increased due to the cross modulationproducts in the output.

By automatically selecting a dc potential the sensitivity can beoptimized. This may sometimes require the use of a calibrating dose ofanalyte for each measurement (See Example 2 as a means of minimizingdrift.)

The present invention also provides an improved method for detectingresponse to an analyte in which a signal may derived by altering andmonitoring dc bias potential, while analyte is binding to the channelsduring the biosensor gating event, either to maintain the admittanceconstant preferably at the frequency for minimum phase or similarly tomaintain the phase constant preferably at the frequency for minimumphase.

The present invention further provides an improved method for detect theelectrode response to analyte in which the signal response is optimizedby automatically altering the dc bias potential to obtain maximumsensitivity or minimum drift.

In order that the nature of the present invention maybe more clearlyunderstood the invention will now be described by way of non-limitingexample.

EXAMPLE 1

On a clean glass or plastic slide, an adhesion layer of chromium (50angstrom) followed by a gold layer (200-2000 angstroms) is evaporated.The freshly evaporated sold coated electrode is taken and immediatelyimmersed in an ethanolic solution of linker lipid (FIG. 1) (300 ul of 10mM), the disulfide of mercaptoacetic acid (150 ul of 10 mM), linkergramicidin (FIG. 2) (150 ul of 0.01 mg/ml, membrane spanning lipid C(FIG. 3) (2.25 ul of 0.1 mM) and membrane spanning lipid D (FIG. 3) (45ul of 1 mM) in ethanol (50 ml). The gold coated electrode is leftimmersed in the solution for 5-60 minutes. rinsed with ethanol andassembled into a Teflon slide assemble holder such that an electrodesurface is defined by a circular Teflon well pressed onto the goldelectrode. The Teflon well forms a tight, water impermeable seal at theelectrode perimeter. This procedure forms the first layer of the bilayersensor membrane and may be stored in ethanol, glycerol, ethylene glycolor other alcohol for several months. Formation of the second layer ofthe bilayer membrane is carried out by addition of 5 ul of a solutioncontaining 14 mM of diphytlayl phosphatidyl choline/glyceryl diphytanylether (7:3 ratio), biotinylated gramicidin (FIG. 4) in a ratio of100.000:1 (total lipid):gramicidin. The well assembly was then rinsedtwice with phosphate buffered saline (PBS) resulting the formation ofthe second lipid layer of the bilayer sensing membrane. The wellassembly holds approximately 150 ul of PBS. Into this 150 ul of PBS inthe well is placed a counter electrode, a connection is made betweenthis counter electrode and the impedance bridge measure apparatus. Tocomplete the electrical circuit, the other connection on is made betweenthe gold electrode and the impedance bridge. In order to control the dcpotential offset a reference electrode is inserted into the well alsocontacting the PBS solution and the potential is controlled such thatthe gold electrode potential may be varied. The apparatus needed to makesuch three terminal measurements are known to those skilled in the art.Alternatively, the dc offset may be varied by changing the metal typewhich makes up the counter electrode. This sets up electrochemicalpotential between the counter electrode and the gold electrode. A dcoffset may also be produced electronically in a two terminal measurementusing the impedance bridge the conduction of the membrane may then bedetermined. Standard Bode plots are shown in FIG. 5. The effect ofchanging the counter electrode material, thus changing the potential, ongramicidin induced membrane conduction is shown. As can be seenstainless steel and titanium counter electrodes produce more conductivemembranes than silver or gold counter electrodes when equivalentmembrane sensor electrodes are measured.

Using a three terminal measurement it was found that the gramicidininduced membrane conduction increases as a negative potential is appliedto the sensor membrane in the range of between 0 mV to −500 mV. FIG. 6shows the effect of Long the potential on gramicidin containingmembranes. As an indication of conduction the frequency at phase minimumis used. The higher the frequency at phase minimum, the more conductivethe membrane. However, on application of a positive potential (0 mV to+50 mV) relative to the gold electrode the gramicidin induced membraneconduction decreased, such that at +200 mV the membrane was ionicallyinsulating.

It is believed that using counter electrode metals such as stainlesssteel or titanium places a dc offset of between −150 mV to 400 mV on thegold electrode relative to the counter electrode. It has been furtherfound that thereproducibility in terms of conduction for a particularconcentration of ionophore in the membrane has been improved fromcoefficients of variation (cv's) of 30-60% using silver counterelectrodes to cv's of 10-15% using stainless steel electrodes.

Similar effects were found if ionophores such as valinomycin were usedinstead of the gramicidin derivatives.

EXAMPLE 2

A membrane was formed as described above in Example 1. A dc offsetacross the biosensor membrane was established using a three terminalvoltage clamp with a platinum counter electrode, a silver chloridereference electrode and a gold sensing electrode.

FIG. 10 shows the effect of varying the dc offset on the drift in thebiosensor output. The output signal was the frequency at minimum phase.The graph Y axis shows the rate of the frequency at minimum phasedivided by the initial frequency at minimum phase.

The RC network in FIG. 10 is a representation of the passive electricalproperties of the sensor membrane. This model consists of two capacitorsconnected in series, one with a value of 0.1 microFarad and the secondwith a value of 0.01 microFarad and a resistor of about 300 kilOhmconnected in parallel with the 0.01 microFarad capacitor. When thisnetwork was connected to the measuring apparatus the intrinsic drift inthe apparatus was found to be negligible as is indicated in. FIG. 10.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described, the present embodiments are,therefore, to be considered in all respect as illustrative and notrestrictive.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the structural formula for a linker lipid.

FIG. 2 depicts the structural formula for the linker Gramicid.

FIG. 3 depicts the structural formulae for membrane spanning lipids.

FIG. 4 depicts the structural formula for Biotinylated Gramicidin.

FIG. 5A is a graph of impedance as a function of frequency for silvercounter electrodes.

FIG. 5B is a graph of phase as a function of frequency for silvercounter electrodes.

FIG. 5C is a graph of impedance as a function of frequency for goldcounter electrodes.

FIG. 5D is a graph of phase as a function of frequency for gold counterelectrodes.

FIG. 5E is a graph of impedance as a function of frequency for stainlesssteel counter electrodes.

FIG. 5F is a graph of phase as a function of frequency for stainlesssteel counter electrodes.

FIG. 5G is a graph of impedance as a function of frequency for titaniumcounter electrodes.

FIG. 5H is a graph of phase as a function of frequency for titaniumcounter electrodes.

FIG. 6 is a graph of conduction at minimum phase as a function ofpotential for a three terminal bridge.

FIG. 7 depicts the structure formulae for GDPE and DPEPC.

FIG. 8A is a schematic diagram of a portion of the circuit of thepresent invention.

FIG. 8B is a schematic diagram of a further portion of the circuit ofthe present invention.

FIG. 9A is a schematic diagram of the logic components of the presentinvention.

FIG. 9B is a schematic diagram of the electrical components of thepresent invention.

FIG. 10 is a representation of the passive electrical properties of thesensor membrane.

What is claimed is:
 1. An improved membrane based biosensor comprising alipid membrane incorporating ionophores, the conductivity of the lipidmembrane being dependent on the presence or absence of an analyte, areference electrode, a sensing electrode onto which is deposited thelipid membrane such that a functional reservoir exists between the lipidmembrane and the sensing electrode, the improvement comprising includingin the biosensor means to apply a dc electrical potential offset to thesensing electrode relative to the reference electrode, said dcelectrical potential offset being produced by a counter electrode.
 2. Animproved membrane based biosensor according to claim 1, wherein themeans to apply a dc electrical potential is a means capable of applyinga dc electrical potential of between +500 mV to −500 mV the sensingelectrode.
 3. An improved membrane based biosensor according to claim 1,wherein the electrochemical potential between the counter electrode andthe sensing electrode produces an electrical potential of between 0 to−500 mV, with the sensing electrode being at the negative potential. 4.An improved membrane based biosensor according to claim 1, wherein thecounter electrode is made from stainless steel.
 5. An improved membranebased biosensor according to claim 1, wherein the counter electrode ismade from titanium.
 6. An improved membrane based biosensor according toclaim 1, wherein the counter electrode is made from metallic elementselected from the group consisting of silver, gold, platinum, palladium,copper, chromium, or molybdenum.
 7. An improved membrane based biosensoraccording to claim 1, wherein the counter electrode is made from a metalthat is capable of being deposited in a thin film onto a plastic, glassor silicon substrate, said metal being stable for at least 30 minutes inaqueous solution and sets up the appropriate electrochemical potentialrelative to the sensing electrode on addition of an aqueous solution. 8.An improved membrane based biosensor according to claim 1, wherein thecounter electrode is an electrochemically neutral metal relative to thesensing electrode and the dc electrical potential of between +500 mV to−500 mV is created by electronic means.
 9. An improved membrane basedbiosensor according to claim 1, wherein the counter electrode producesan electrochemical potential relative to the sensing electrode which isenhanced or negated or reversed using a dc electrical potential createdby electronic means to give a potential of between +500 mV to −500 mV.10. An improved membrane based biosensor according to claim 1, whereinthe dc offset potential at the sensing electrode, onto which isdeposited a lipid membrane, is controlled using a three terminalmeasurement, wherein the impedance measurement is made between thecounter electrode and the working electrode which is the sensingelectrode and where the dc offset potential is controlled by a referenceelectrode to be between +500 mV to −500 mV as required.
 11. An improvedmembrane based biosensor according to claim 10, wherein the sensingelectrode comprises a metal.
 12. An improved membrane based biosensoraccording to claim 11, wherein the metal used for the sensing electrodeis a layer of freshly evaporated, sputtered, plasma etched or ion beammilled gold.
 13. An improved membrane based biosensor according to claim1, wherein the lipid membrane comprises a first layer of linker lipid(FIG. 1), the disulfide of mercaptoacetic acid, linker gramicidin (FIG.2), membrane spanning lipid C (FIG. 3) and membrane spanning lipid D(FIG. 3).
 14. An improved membrane based biosensor according to claim13, wherein the lipid membrane comprises a second layer of diphytanylphosphatidyl choline, glycerol, diphytanyl ether, and biotinylatedgramicidin (FIG. 4).
 15. An improved membrane based biosensor accordingto claim 14, wherein the said second layer contains at least aproportion of a phosphatidyl choline, or phosphatidyl ethanolamine orphosphatidic acid lipid.
 16. An improved membrane based biosensoraccording to claim 14 wherein said second layer contains at least aproportion of a charged lipid.
 17. An improved membrane based biosensoraccording to claim 11, wherein the lipid membrane is a monolayer.
 18. Animproved method detecting the presence or absence of an analyte in asample using a membrane based biosensor comprising a lipid membraneincorporating ionophores, the conductivity of the lipid membrane beingdependent on the presence or absence of the a reference electrode, asensing electrode onto which is deposited the lipid membrane such that afunctional reservoir exists between the lipid membrane and the sensingelectrode, the improvement comprising applying a dc electrical potentialoffset to the sensing electrode relative to the reference electrode,said dc electrical potential offset being produced by a counterelectrode.
 19. An improved method according to claim 18, wherein a dcelectrical potential of between +500 mV to −500 mV is applied to thesensing electrode.
 20. An improved method according to claim 18, whereinthe electrochemical potential between the counter electrode and thesensing electrode produces an electrical potential of between 0 to −500mV, with the sensing electrode being at the negative potential.
 21. Animproved method according to claim 18, wherein the counter electrode ismade from stainless steel.
 22. An improved method according to claim 18,wherein the counter electrode is made from titanium.
 23. An improvedmethod according to claim 18, wherein the counter electrode is made frommetallic element selected from the group consisting of silver, gold,platinum, palladium, copper, chromium or molybdenum.
 24. An improvedmethod according to claim 18, wherein the counter electrode is made froma metal that is capable of being deposited in a thin film onto aplastic, glass or silicon substrate, said metal being stable for atleast 30 minutes in aqueous solution and sets up the appropriateelectrode chemical potential relative to the sensing electrode onaddition of an aqueous solution.
 25. An improved membrane basedbiosensor according to claim 18, wherein the counter electrode is anelectrochemically neutral metal relative to the sensing electrode andthe dc electrical potential of between +500 mV b −500 mV is created byelectronic means.
 26. An improved membrane based biosensor according toclaim 18, wherein the counter electrode produces an electrochemicalpotential relative to the sensing electrode which is enhanced or negatedor reversed using a dc electrical potential created by electronic mansto give a potential of between +500 mV to −500 mV.
 27. An improvedmembrane based biosensor according to claim 18, wherein the dc offsetpotential at the sensing electrode, onto which is deposited a lipidmembrane, is controlled using a three terminal measurement, wherein theimpedance measurement is made between the counter electrode and theworking electrode which is the sensing electrode and where the dc offsetpotential is controlled by a reference electrode to be between +500 mVto −500 mV as required.
 28. An improved membrane based biosensoraccording to claim 18, wherein the sensing electrode comprises metal.29. An improved method according to claim 28, wherein the metal used forthe sensing electrode is a layer of freshly evaporated, sputtered,plasma etched or ion beam milled old.
 30. An improved method accordingto any one of claims 18, wherein the lipid membrane comprises a firstlayer of linker lipid (FIG. 1), the disulfide of mercaptoacetic acid,linker gramicidin (FIG. 2), membrane spanning lipid C (FIG. 3) andmembrane spanning lipid D (FIG. 3).
 31. An improved method accordingclaim 30, wherein the lipid membrane comprises a second layer diphytanylphosphatidyl choline, glycerol diphytanyl ether, and biotinylatedgramicidin (FIG. 4).
 32. An improved membrane based biosensor accordingto claim 31, wherein the said second layer contains at least aproportion of a phosphatidyl choline, or phosphatidyl ethanolae orphosphatidic acid lipid.
 33. An improved membrane based biosensoraccording to claim 31, wherein said second layer contains at least aproportion of a charged lipid.
 34. An improved membrane based biosensoraccording to claim 18, wherein the sensing membrane is a monolayer.